Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus in man. It is rarely pathogenic in healthy adults but is associated with several diseases in immunocompromised individuals (such as HIV-infected people and transplant recipients). Furthermore, HCMV is the most common cause of congenital infection in humans. Intrauterine primary infections are second only to Down's syndrome as a known cause of mental retardation. Less severe complications are the result of secondary infections. As infections are either asymptomatic or accompanied by symptoms that are not specific of HCMV (such as fever and leukopenia), laboratory techniques are the sole means of diagnosing acute HCMV infection. Diagnosis of HCMV infection can be obtained by direct demonstration of the virus or virus components in pathological materials or indirectly through serology 111. Diagnosis of primary HCMV infection is exclusively accomplished by serological methods, i.e. demonstration of the appearance of antibodies to HCMV in a previously seronegative subject. HCMV-specific IgM is a sensitive and specific indicator of primary HCMV infection in immunocompetent subjects while it is very often produced during active viral reactivation in transplant recipients [2-4]. However its detection varies widely and a very poor agreement has been found among the results obtained with different commercial kits [5].
From EP 262531 there are known immunogenic portions of HCVM structural phosphoprotein of 150 kD, encoded by the gene localized in the Hind III-Y/N fragment of the viral genome; according to said European patent, such immunogenic portions of pp150 are encoded, in particular, by an EcoRI-PstI fragment of approximately 1.5 kb, localized inside the region of EcoRi-Y fragment from HCMV genome of AD169 strain. Subsequent and more exhaustive studies have however shown the afore deductions to be incorrect, in that (FIG. 1), protein pp150 is shown to be encoded by UL32, much longer than 1.5 kb and, anyway, quite outside any such EcoRI-PstI fragment, as may be defined within the EcoRI-Y fragment of the AD169 strain. Furthermore, the EcoRI-Y fragment is wholly outside (in particular, to the side, towards the NH.sub.2 -terminus) of Hind III-Y/N fragment. According to the Applicants, therefore, there is a correlation between the immunogenic properties shown by the proteic material referred to in the afore European patent (which, however, providing an incorrect identification of the polypeptide, causes it to remain substantially undetermined) and the inclusion into the peptide of epitope A1C2, encoded by UL32 nucleotides 1783 to 1842, running 5'.fwdarw.3', i.e., of the region corresponding to amino acids 595 to 614 of ppUL32, whose identification is posterior (Novak J. P. et al. 1991. Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides. J. Gen. Virol. 72; 1409-1413).
Antigenic materials composed of single well characterized viral proteins, or portions of them, produced via molecular biology or peptide chemistry have proven to be promising tools in improving serological diagnosis [6-14]. The analysis of the humoral immune response elicited during natural infection has repeatedly shown that the basic phosphoprotein of 150 kD encoded by UL32 (ppUL32) [15] and localized in the viral tegument is highly immunogenic and is recognized by sera from nearly 100% of the HCMV-seropositive subjects tested [6, 16]. In this molecule at least two epitopes have been identified and shown to react efficiently with human immunoglobulins. In particular, the analysis of several ppUL32 fusion proteins showed that a region localized in the sequence between aa 1006 and aa 1048, inclusive, read in 5'.fwdarw.3' direction, of the molecule (aa 1006-1048) reacts with more than 80% of IgM-positive sera [9,11]. When chemically synthesized, another region localized between aa 595 and 614 gave a positive reaction with almost 100% of IgG-positive sera [17]. When expressed as recombinant protein this region has proven to react very efficiently also with HCMV-specific IgM [12]. The two coding regions were recently fused together and shown to produce a double epitope fusion protein which can replace the entire pp150 molecule in its IgM-binding ability [12].
Another HCMV protein which reacts very well with IgM is the non structural DNA-binding phosphoprotein of 52 kD [18] encoded by viral gene UL44 (ppUL44) [7, 11]. The COOH part of the molecule shows efficient IgM binding and does not contain relevant amino acid sequences cross-reacting with the homologous protein of other members of the Herpesviridae family (that are mainly present in the NH.sub.2 half) [19]. The COOH half of this protein was expressed and tested with human sera. The DNA sequence expressing this region has been in fact inserted into plasmid p-ROS (which carries a truncated sequence of LacZ) at site SmaI and cloned into E.coli, thus obtaining a fusion protein H10, which has yielded good results [21].
Besides ppUL32 and ppUL44, two other HCMV proteins are well known: the major matrix phosphoprotein of 65 kD encoded by viral gene UL83 (ppUL83) and the assembly phosphoprotein of 38 kD encoded by the viral gene UL80a (ppUL80a). The first is known to induce a strong IgM response and antibodies reacting exclusively to this protein were described during primary infection [7]. Likewise, the IgM to this second protein are very often the first marker to be detected in HCMV seropositive transplanted patients undergoing a viral reactivation [Kraat et al. 1995, manuscript submitted]. Moreover, some of the Applicants observed a strong IgM reactivity to this protein in congenitally infected newborns (Lazzarotto and Landini, unpublished observation).
However, the above studies have not been successful, so far, in producing such recombinant proteic material, as are actually capable of replacing the virus or virus components (purified virions) in the diagnostic kits currently in use.